CN109541231A - A kind of detection antibody of HCV kit and preparation method thereof - Google Patents
A kind of detection antibody of HCV kit and preparation method thereof Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Abstract
The invention discloses a kind of detection antibody of HCV kits and preparation method thereof, are grouped as by following group: Streptavidin magnetic microsphere, negative controls, positive reference substance, IgG1 antibody marker, biotinylated antigen, analysis buffer, cleaning solution, enhancement solution and RFID card in conjunction with lanthanide series.This method is based on magnetic microsphere and combines with Timed-resolved fluoroimmunoassay, both it is longer the physical sorption reaction time of ELISA Plate had been overcome, the slower drawback of testing result, the reaction time is greatly shortened, while also having that time resolution detection technique accuracy height, high sensitivity, high specificity, the range of linearity is wide, detection is stable and convenient advantage.Magnetic microsphere is coated with corresponding antigen or antibody and greatly increases the contact surface area of immune response because of the characteristic of magnetic microsphere solid, to greatly shorten detection time, result can be detected in 30 minutes.
Description
Technical field
The present invention relates to a kind of detection antibody of HCV kits and preparation method thereof, and in particular to one kind is based on
Magnetic microsphere combines the preparation method of detection antibody of HCV kit with Timed-resolved fluoroimmunoassay.
Background technique
Hepatitis C Virus (HCV) particle spherical in shape, has lipidic shell, cyst membrane and spinous process structure outside, inside has by core egg
The nucleocapsid of white and nucleic acid composition.5 ' noncoding region of HCV genome is conservative, in the PCR primer designed for diagnosis HCV infection
When, this section is first choice.In addition the gene regions NS3 coding helicase and albumen in non-structural protein white area (NS2, NS3, NS4, NS5)
Enzyme, NS3 albumen have it is strongly immunogenic, can stimulate body generate antibody, have important value in clinical diagnosis.Anti-HCV is not
Protection antibody is HCV infection mark.Most of HCV infections are all hidden in acute stage and chronic infection early symptom, chronicity
Rate is 60% to 85%, wherein again more with older, male office.
At present hepatitis C virus Serology test have enzyme linked immunological (ELISA), colloidal gold method, chemiluminescence (CLIA),
Electrochemical luminescence (ECL), time-resolved fluoroimmunoassay (TRFIA) etc..ELISA is existing application as semi-quantitative detection method
Most wide detection method, but its sensitivity, linear extent range are not up to higher level, it is difficult to adapt to the need of market development
It asks.Colloidal gold method and ELISA have the shortcomings that identical.Though and CLIA and ECL high sensitivity but to there is detection device cost high
It is high, and corresponding marker research and development threshold is high, domestic disadvantage that can be under one's control equally also limits at home in short-term
Popularization.Time-resolved fluoroimmunoassay (TRFIA) sensitivity can reach the consistent level of CLIA, not only detection device cost
It is lower, and the relevant technologies are quite mature at home.Conventional temporal resolved detection is still with the physical absorption of blank ELISA Plate
Main, detection process takes a long time.Shorten the reaction time, reduce testing cost, improves detection sensitivity and the range of linearity, will have
Good market prospects.
Summary of the invention
It is provided based on this it is an object of the invention to overcome the prior art and the high disadvantage of the relevant technologies testing cost
A kind of preparation method of detection antibody of HCV kit and the kit.
To achieve the above object, the present invention adopts the following technical scheme that:
A kind of detection antibody of HCV kit, is grouped as by following group: Streptavidin (SA) magnetic microsphere, feminine gender
Reference substance, positive reference substance, the IgG1 antibody marker in conjunction with lanthanide series, biotinylated antigen, analysis buffer, concentration
Washing lotion (cleaning solution) and enhancement solution, RFID card.
Preferably, the IgG1 antibody marker in conjunction with lanthanide series, lanthanide series therein are merged with IgG1
Protein antibodies are combined by intermediate chelating agent, and lanthanide series includes but is not limited to europium (EU), samarium (Sm), chelating agent include but
It is not limited to isothiocyanic acid phenyl-EDTA, isothiocyanic acid benzyl-DTTA, P- isothiocyanatobenzyl-DTTA, diethylenetriamine five
Acetic acid aminophenyl-EDTA.
Preferably, the biotin antigen is by HCV recombinant antigen (containing NS3, CORE) and HCV biotinylated antigen
(containing NS3, CORE) is made after mixing by certain dilution ratio.
The present invention also provides a kind of methods using mentioned reagent box detection anti-HCV, and described method includes following steps:
(1) IgG1 antibody marker is diluted to working solution with analysis buffer；
(2) working solution is diluted to by washing lotion is concentrated with purified water；
(3) it is added in reaction cup after mixing the SA magnetic microsphere of the working concentration diluted；
(4) sample to be examined or negative controls, positive reference substance are added in above-mentioned reaction cup；
(5) biotinylated antigen is added in reaction cup, is incubated at room temperature；
(6) use the cleaning liquid reaction cup in (2) that magnetic is added to wash after being incubated for；
(7) after washing, marker working solution in (1) is added, is incubated at room temperature；
(8) use the cleaning liquid reaction cup in (2) that magnetic is added to wash after being incubated for；
(9) after washing, enhancement solution is added and is incubated for；
(10) after being incubated for, the fluorescent collecting for carrying out corresponding wavelength is detected and is analyzed.
Preferably, for the present invention in order to further reduce manual steps, self-produced SmartTRF grinds certainly in cooperation company
The relevant parameter of detection method, operating procedure are all copied to RFID card by complete series Immunofluorescence test equipment
In.In actual mechanical process, it is only necessary to RFID card is adapted to above-mentioned Immunofluorescence test equipment can be automatically finished it is above-mentioned
The operating procedure of experiment.RFID(Radio Frequency Identification) technology, also known as radio frequency identification are one
The kind communication technology can be identified specific objective by radio signals and read and write related data, without identifying system and specific mesh
Mechanical or optical contact is established between mark.
The present invention also provides a kind of preparation methods for detecting antibody to hepatitis C kit, and the method includes walking as follows
It is rapid:
(1) it after cleaning, buffering displacement SA magnetic bead, is diluted to working concentration and SA magnetic microsphere is made；
(2) drop matches HCV recombinant antigen and HCV bioid antigen, and the two is mixed and made into kit bioid with concentration by best drop
Antigen；
(3) negative controls, positive reference substance are prepared；
(4) the IgG1 antibody marker of europium label is prepared；
(5) analysis buffer, concentration washing lotion and enhancement solution are prepared；
(6) each component is dispensed respectively into corresponding storage container；
(7) RFID card duplicate copy；
(8) coding, labelling；
(9) it is assembled into finished product kit.
Compared with prior art, the beneficial effects of the invention are that:
This method is based on magnetic microsphere and combines with Timed-resolved fluoroimmunoassay, has both overcome the physical adsorption reaction of ELISA Plate
Time is longer, and the slower drawback of testing result greatly shortens the reaction time, while also having time resolution detection technique accuracy
Height, high sensitivity, high specificity, the range of linearity are wide, detection is stable and convenient advantage.Magnetic microsphere be coated with corresponding antigen or
Antibody greatly increases the contact surface area of immune response because of the characteristic of magnetic microsphere solid, so that detection time is greatly shortened,
Result can be detected in 30 minutes.In addition, being no longer limited by the frame limit of traditional ELISA Plate due to the fluid behaviour of magnetic microsphere
System, can be completed detection, while can also reduce the volume and cost of detecting instrument equipment in arbitrary reaction cup and small test tube
To meet the full-automatic detection demand in two, three line cities, also big flux inspection can be carried out as ELISA Plate, the reflective detection of chemistry
It surveys, meets the full-automatic detection demand in a line city.
Detailed description of the invention
Fig. 1 is to be used to store Streptavidin (SA) magnetic microsphere, europium label knot using detection kit of the present invention
The IgG1 antibody marker of conjunction, biotinylated antigen, analysis buffer, cleaning solution and enhancement solution reagent strip vertical view signal
Figure.
Fig. 2 is to be used to store Streptavidin (SA) magnetic microsphere, europium label knot using detection kit of the present invention
The IgG1 antibody marker of conjunction, biotinylated antigen, analysis buffer, cleaning solution and enhancement solution reagent strip stereochemical structure mark
It is intended to schematic diagram.
Specific embodiment
The present invention is further illustrated with attached drawing with reference to embodiments, and it is special that technology of the invention is better described
Point, technical solution.Following embodiment does not cause any restrictions to the present invention.SA magnetic microsphere derives from following embodiment
JSR company；Europium label is purchased from Wallac company, Finland；Antibody of HCV National reference is examined by Chinese food drug
Research institute's offer is provided；Contrast agents box is Abbott Laboratories' anti-HCV detection kit (chemiluminescence particulate immune detection
Method).
Embodiment 1
A kind of detection antibody of HCV kit, the kit include: Streptavidin (SA) magnetic microsphere, feminine gender
Reference substance, positive reference substance, the IgG1 antibody marker in conjunction with lanthanide series, biotinylated antigen, analysis buffer, concentration
Washing lotion and enhancement solution, RFID card.
The present invention also provides the preparation methods of above-mentioned detection antibody of HCV kit, and the method includes as follows
Step:
(1) it prepares Streptavidin (SA) magnetic microsphere: 3 μm of Streptavidins (SA) magnetic microsphere is used into 25mM PH7.0
Tris-HCL buffer clean 4 times repeatedly on magnetic frame after high concentration save it is stand-by；It, will be high after the completion of other components preparation
Concentration Streptavidin (SA) magnetic microsphere carries out drop by different use concentration and matches, and final magnetic microsphere is 100 μ g/ using concentration
Ml, liquid volume added are 50 holes μ L/.
(2) prepare europium label IgG1 antibody marker: by IgG1 antibody be placed in molecular cut off be 10000 ultrafiltration from
In heart pipe, 10000rpm is centrifuged 7~10min, discards filtrate.Add 9.6 carbonate buffer solution 10000rpm of 0.05M PH from
7~10min of the heart 2~3 times repeatedly, centrifuge tube filter membrane reversion 3000rpm is centrifuged 6min, collects 200 μ L solution being finally concentrated.
And by it and in advance with the solvent DTTA-EU of carbonate buffer solution3+Mixing, IgG1 antibody and europium mass ratio are 3:1,2~8 DEG C of vibrations
Swing mixing 48 ± 2 hours.The Sephadex that label solution is balanced through 7.8 Tris-Hcl buffer of 0.05M PHTMG-50 gel
Column (1.0*50cm) chromatographic purifying monitors in A280 and collects first peak.By the IgG1 antibody of the europium being collected into label with 0.2%
7.8 Tris-Hcl buffer of 0.05M PH is diluted to 1/20 times of optium concentration, dispenses to 1.0mL/ bottles.
(3) biotinylated antigen is prepared: by HCV recombinant antigen and the Tris- of HCV bioid antigen 25mM PH7.0
HCL buffer press respectively 1/1500,1/1000 dilution mixing biotinylated antigen.
(4) negative controls, positive reference substance are prepared: using 5% BSA, 0.5% Tween-20,0.02%
Negative controls, the positive are made in c-hepatitis antibody strong positive serum by 7.8 0.02M Tris-HCl buffer of Proclin300, pH
Reference substance.
(5) it prepares analysis buffer: containing Tween-20, Proclin300, EDTA, BSA, Tris-HCl buffer, dividing
It is filled to 30~40mL/ bottles.
(6) preparation concentration washing lotion: containing Tween-20, Proclin300, Tris-Hcl buffer, dispense to 30~
40mL/ bottles.
(7) it prepares enhancement solution: containing sodium acetate, β-NTA, TOPO, glacial acetic acid, dehydrated alcohol, Triton X-100, dividing
It is filled to 30~40mL/ bottles.
(8) it prepares RFID card: blank RFID card is subjected to relative parameters setting by detection method in the present embodiment of the present invention；
(9) coding, labelling assemble kit.
The present invention also provides the detection method of above-mentioned detection antibody of HCV kit, the method concrete operations
It is as follows:
(1) reagent prepares
Kit restores in being placed at room temperature for room temperature；
The IgG1 antibody marker of europium label is diluted 20 times to working solution using analysis buffer, is mixed stand-by；
Cleaning solution: purified water is added by 1:25 in concentration washing lotion and is diluted to work cleaning solution；
Upright light rolling SA magnetic microsphere, mixes stand-by before experiment；
(2) experimental implementation
The SA magnetic microsphere for drawing 50 μ L mixing is added in reaction cup；
Sample to be tested or negative controls, 100 μ L of positive reference substance are added into each reaction cup；
100 μ L biotinylated antigens are added into each reaction cup again；
It is stored at room temperature incubation 15 minutes；
After incubation, added magnetic cleaning 4 times for the first time using cleaning solution；
After cleaning, the IgG1 antibody marker of 100 μ L europiums label is added into each reaction cup for degaussing, and stationary incubation 15 divides
Clock；
After incubation, use second plus magnetic cleaning 4 times of cleaning solution；
After cleaning, 100 μ L enhancement solutions are added into each reaction cup for degaussing, are stored at room temperature incubation 3 minutes；It is complete in 30 minutes
At fluorescent collecting and carry out data analysis.
In the present embodiment, actual laboratory operating procedures and relevant parameter are copied in matched RFID card in advance, real
It tests after operating process only needs to be ready to by reagent preparation process, RFID card is adapted to fully-automatic equipment can be completed from information
Read, be loaded onto the overall process of detection.
Embodiment 2
A kind of detection antibody of HCV kit, essentially identical with detection kit described in embodiment 1, difference exists
In:
(1) the detection antibody of HCV kit component include: reagent strip, negative controls, positive reference substance,
RFID card.
(2) in the present embodiment, reagent strip is marked by Streptavidin (SA) magnetic microsphere, europium in the detection kit
In conjunction with IgG1 antibody marker, biotinylated antigen, analysis buffer, cleaning solution and enhancement solution dispense it is corresponding to reagent strip
It is formed after sealer in hole.Wherein cleaning solution is that concentration is washed in embodiment 1 plus purified water dilutes 25 times and forms；Remaining each component and reality
It applies identical in example 1.
In the present embodiment, just as shown in Figure 1 and Figure 2, each hole bit function of reagent strip is described as follows in the detection kit:
Reagent strip is from left to right arranged successively, and title is followed successively by the 1st~13 hole.1st, 2 holes are instrument connection, the 3rd, 4 holes be
Fluorescent marker hole, the 5th hole be analysis buffer hole, the 6th, 7 holes be cleaning fluid apertures, the 8th, 9 be Sample Dilution fluid apertures, the 12nd for increase
Strong fluid apertures, the 10th, 11,13 be preparation hole.1st and 2 holes are the reacting hole storing magnetic microsphere and being immunoreacted, and maximum can
Storage liquid volume is 800 μ L；3rd, 4 holes can be disassembled into from entire reagent strip and be independent component, convenient for fluorescence mark
Note object carries out packing storage.3rd, 4,5 holes can store maximum liquid volume be 400 μ L；6th, 7 holes can store maximum liquid
Volume is 3000 μ L；It is 400 μ L that 8th ~ 12 hole, which can store maximum liquid volume,；13rd hole can store maximum liquid volume
600μL。
In the present embodiment, the reagent strip in the detection kit is made after carrying out sealer as follows:
IgG1 antibody marker, the 150 μ L biotinylations that 300 μ L Streptavidin (SA) magnetic microspheres, 50 μ L europiums are marked are anti-
Original, 200 μ L analysis buffers, 3000 μ L cleaning solutions, 200 μ L enhancement solutions are dispensed respectively to the 1st of reagent strip the, 3,10,5,6,12
In hole, the reagent strip is made after sealer coding.
The present invention also provides the preparation methods of above-mentioned detection antibody of HCV kit, and examination is obtained in the above describe manner
After agent item, kit is constituted with negative controls, positive reference substance, RFID card.In addition to each component dispenses mode and storage
Container is different outer, remaining is in the same manner as in Example 1.
The present invention also provides the detection methods of above-mentioned detection antibody of HCV kit, it is only necessary to by mentioned reagent
Item is inserted into the reagent clamp bar slot of SmartTRF serial equipment after gently shaking mixing, and equipment reads RFID card relevant information can entirely certainly
It is dynamic to complete detection process.The related information parameters and detecting step of RFID card are in the same manner as in Example 1.
Embodiment 3
The performance evaluation of detection antibody of HCV kit of the present invention:
By the kit and detection method prepared in embodiment, third type liver of the detection buying from National Institute for Food and Drugs Control
Scorching antiviral antibody National reference, and collect the clinic from Abbott Laboratories, hospital Architect i2000 detection c-hepatitis antibody
262, sample.
Embodiment 1, embodiment 2 detect antibody of HCV National reference, as a result as follows:
(1) negative reference product coincidence rate: 30 parts of negative National reference N1~N30, embodiment 1, embodiment 2 detect coincidence rate
(-/-) it is 30/30；
(2) positive reference product coincidence rate: 30 parts of positive National reference P1~P30, embodiment 1, embodiment 2 detect coincidence rate
(+/+) it is 30/30；
(3) accuracy: accuracy reference material J repeats detection 10 times, and embodiment 1, embodiment 2 detect accuracy C.V≤8.4%；
(4) minimum detectability: L1, L2 detect the positive, L3, L4 detection are feminine gender.
Embodiment 1, embodiment 2 detect 262 clinical samples of Abbott Laboratories' anti-HCV, as a result as follows:
The comparison of 1 embodiment of table, 1 clinical sample
Detection kit described in the embodiment of the present invention 1 detects antibody of HCV and contrast agents negative sample coincidence rate
100%, positive sample coincidence rate 100%.
The comparison of 2 embodiment of table, 2 clinical sample
Detection kit described in the embodiment of the present invention 2 detects antibody of HCV and contrast agents negative sample coincidence rate
100%, positive sample coincidence rate 100%.
What concentration washing lotion (cleaning solution) referred in embodiment 1 is the high concentration cleaning solution to be diluted to working solution；Cleaning
What liquid referred in example 2 is the working solution for not needing any processing.
It should be understood that detecting the detection method and preparation method of antibody of HCV kit in embodiment 1
It is to be invented to meet big flux testing goal, instrument and equipment volume is larger and cost is relatively high, in order to further full
The detection demand in two, three line cities of foot, correspondingly, we have further made some detection methods on the basis of embodiment 1
And the modification on reagent box preparation method, as in embodiment 2.
It should be understood that difference of the present invention in example 2 with detection method and preparation method in embodiment 1 exists
In embodiment 2 is the IgG1 antibody marker for combining Streptavidin (SA) magnetic microsphere, the europium label in embodiment 1, life
Object element antigen, analysis buffer, cleaning solution and enhancement solution dispense simultaneously into special reagent strip (as shown in Figure 1 and Figure 2), because
The component of kit only has reagent strip, RFID card, negative controls, positive reference substance in this embodiment 2.Wherein RFID card, yin
It is consistent in property reference substance, positive reference substance and embodiment 1.
It should be understood that the detection method of the present invention in example 2 be by reagent strip be inserted into detection device after, will be corresponding
RFID card be adapted to equipment, equipment whole-course automation operating procedure is consistent with embodiment 1；
It should be understood that the preparation method of the detection kit of the present invention in example 2 is with the difference in embodiment 1,
Step (6) becomes " dispensing each component to the corresponding aperture of reagent strip respectively, sealer coding after having dispensed ".
Claims (5)
1. a kind of detection antibody of HCV kit, it is characterised in that be grouped as by following group: Streptavidin is magnetic
Microballoon, negative controls, positive reference substance, the IgG1 antibody marker in conjunction with lanthanide series, biotinylated antigen, analysis are slow
Fliud flushing, cleaning solution, enhancement solution and RFID card.
2. detection antibody of HCV kit as described in claim 1, which is characterized in that described and lanthanide series
In conjunction with IgG1 antibody marker, lanthanide series therein and IgG1 fusion protein antibody are the lanthanums by conjunction with intermediate chelating agent
Series elements are europium or samarium, and chelating agent is isothiocyanic acid phenyl-EDTA, isothiocyanic acid benzyl-DTTA, P- isothiocyanatobenzyl-
DTTA or diethylene triamine pentaacetic acid aminophenyl-EDTA.
3. detection antibody of HCV kit as described in claim 1, which is characterized in that the biotin antigen
It is made after being mixed by HCV recombinant antigen with HCV biotinylated antigen.
4. detection antibody of HCV kit as described in claim 1, which is characterized in that the biotin antigen
It is by the Tris-HCL buffer of HCV recombinant antigen and HCV bioid antigen 25mM pH7.0 respectively by 1/1500,1/1000
Dilution mixing biotinylated antigen.
5. the preparation method of detection antibody of HCV kit described in claim 1, it is characterised in that including as follows
Step:
(1) it after cleaning, buffering displacement SA magnetic bead, is diluted to working concentration and Streptavidin magnetic microsphere is made；
(2) drop matches HCV recombinant antigen and HCV bioid antigen, is mixed and made into kit bioid antigen；
(3) negative controls, positive reference substance are prepared；
(4) the IgG1 antibody marker of europium label is prepared；
(5) analysis buffer, concentration washing lotion and enhancement solution are prepared；
(6) each component is dispensed respectively into corresponding storage container；
(7) RFID card duplicate copy；
(8) coding, labelling；
(9) it is assembled into finished product kit.
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| CN201811329848.3A CN109541231A (en) | 2018-11-09 | 2018-11-09 | A kind of detection antibody of HCV kit and preparation method thereof |
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| CN201811329848.3A CN109541231A (en) | 2018-11-09 | 2018-11-09 | A kind of detection antibody of HCV kit and preparation method thereof |
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Application publication date: 20190329 |